Proteins have become commercially important as drugs that are generally called “biologicals”. One of the greatest challenges is the development of cost effective and efficient processes for purification of proteins on a commercial scale. While many methods are now available for large-scale preparation of proteins, crude products, such as cell culture supernatants, contain not only the desired product but also impurities, which are difficult to separate from the desired product. Although cell culture supernatants of cells expressing recombinant protein products may contain less impurities if the cells are grown in serum-free medium, the host cell proteins (HCPs) still remain to be eliminated during the purification process. Additionally, the health authorities request high standards of purity for proteins intended for human administration.
A number of chromatographic systems are known that are widely used for protein purification.
Ion exchange chromatography systems are used for separation of proteins primarily on the basis of differences in charge.
Anion exchangers can be classified as either weak or strong. The charge group on a weak anion exchanger is a weak base, which becomes de-protonated and, therefore, loses its charge at high pH. DEAE-sepharose is an example of a weak anion exchanger, where the amino group can be positively charged below pH ˜9 and gradually loses its charge at higher pH values. Diethylaminoethyl (DEAE) or diethyl-(2-hydroxy-propyl)aminoethyl (QAE) have chloride as counter ion, for instance. A strong anion exchanger, on the other hand, contains a strong base, which remains positively charged throughout the pH range normally used for ion exchange chromatography (pH 1-14). Q-sepharose (Q stands for quaternary ammonium) is an example for a strong anion exchanger.
Cation exchangers can also be classified as either weak or strong. A strong cation exchanger contains a strong acid (such as a sulfopropyl group) that remains charged from pH 1-14; whereas a weak cation exchanger contains a weak acid (such as a carboxymethyl group), which gradually loses its charge as the pH decreases below 4 or 5. Carboxymethyl (CM) and sulphopropyl (SP) have sodium as counter ion, for example.
Hydrophobic interaction chromatography (HIC) is used to separate proteins on the basis of hydrophobic interactions between the hydrophobic moieties of the protein and insoluble, immobilized hydrophobic groups on the matrix. Generally, the protein preparation in a high salt buffer is loaded on the HIC column. The salt in the buffer interacts with water molecules to reduce the salvation of the proteins in solution, thereby exposing hydrophobic regions in the protein which are then adsorbed by hydrophobic groups on the matrix. The more hydrophobic the molecule, the less salt is needed to promote binding. Usually, a decreasing salt gradient is used to elute proteins from a column. As the ionic strength decreases, the exposure of the hydrophilic regions of the protein increases and proteins elute from the column in order of increasing hydrophobicity.
Hydrophobic charge induction chromatography (HCIC) is another mode of chromatography based on the pH dependent behavior of heterocyclic ligands that ionize at low pHs. While adsorption on this mode of chromatography occurs via hydrophobic interactions, desorption is facilitated by lowering the pH to produce charge repulsion between the ionizable ligand and the bound protein (e.g. sorbent MEP Hypercel from Biosepra).
Yet a further way of purifying proteins is based on the affinity of a protein of interest to another protein that is immobilized to a chromatography resin. Examples for such immobilized ligands are the bacterial cell wall proteins Protein A and Protein G, having specificity to the Fc portion of certain immunoglobulins. Although both Protein A and Protein G have a strong affinity for IgG antibodies, they have varying affinities to other immunoglobulin classes and isotypes as well.
Affinity chromatography on protein A allows the clearance of more than 99.5% of the impurities such as host cell proteins (HCPs), DNA, viruses, incomplete forms of the antibodies in only one step. However, the major disadvantage of this purification technique is the cost of the resin. It is approximately 30 times more expensive than ion exchange resins and can represent nearly 35% of the total cost of the raw material used for large scale purification. Protein A resin also presents some stability problems as Protein A residues, which are potentially immunogenic, are found in the eluate and need therefore to be cleared. Protein A resin is also difficult to sanitize as the ligand is easily denatured by common sanitization solutions like sodium hydroxide and this represents a major problem in production in the event of contamination as re-use of the resin may be detrimentally affected.
Combinatorial chemistry has enabled the synthesis of a wide variety of ligands which can mimic the action of protein A e.g. the triazine derivatives that mimic the Phe-132, Tyr-133 dipeptide binding site in the hydrophobic core structure of Protein A (marketed as MAbsorbent A1P, A2P, and A3P by Prometic).
A further way of purifying antibodies uses affinity ligands developed by making use of Camelidae heavy chain antibody fragments (CAPTURESELECT products from The Bio Affinity Company).
In the field of antibody purification, Follman and Fahrner (2004) have determined that the same host cell protein removal obtained with a process incorporating Protein A chromatography can be achieved using a process with no affinity chromatography steps. They identified three non-affinity purification processes including hydrophobic interaction chromatography, anion-exchange chromatography and cation-exchange chromatography that remove CHOPs (Chinese Hamster Ovary Cell Proteins) to levels comparable to the traditional Protein A process (J Chromatogr A. 2004. Jan. 23; 1024(1-2):79-85); WO 03/102132A2). They also disclose a method for protein purification that involves the combination of non-affinity chromatography and high performance tangential flow filtration (HPTFF). After a first purification (capture) step on cation exchange chromatography the host cell protein content was about 14,000 ppm.
Antibodies, or immunoglobulins (Igs) consist of light chains and heavy chains linked together by disulphide bonds. The first domain located at the amino terminus of each chain is variable in amino acid sequence, providing the vast spectrum of antibody binding specificities. These domains are known as variable heavy (VH) and variable light (VL) regions. The other domains of each chain are relatively invariant in amino acid sequence and are known as constant heavy (CH) and constant light (CL) regions.
The major classes of antibodies are IgA, IgD, IgE, IgG and IgM; and these classes may be further divided into subclasses (isotypes). For example, the IgG class has four subclasses, namely, IgG1, IgG2, IgG3, and IgG4.
The differences between antibody classes are derived from differences in the heavy chain constant regions, containing between 1 and 4 constant domains (CH1-CH4), depending on the immunoglobulin class. A so-called hinge region is located between the CH1 and CH2 domains. The hinge region is particularly sensitive to proteolytic cleavage; such proteolysis yields two or three fragments depending on the precise site of cleavage. The part of the heavy chain constant region containing the CH2 and CH3 domains, optionally together with the hinge region, is also called the “Fc” part of the immunoglobulin. Antibodies are thus Fc-containing proteins.
Several antibodies that are used as therapeutic proteins are known. Examples for recombinant antibodies on the market are for instance: Abciximab, Rituximab, Basiliximab, Daclizumab, Palivizumab, Infliximab, Trastuzumab, Alemtuzumab, Adalimumab, Cetuximab, Efalizumab, Ibritumomab, Bevacizumab, or Omalizumab.
Another type of Fc-containing proteins are the so-called Fc-fusion proteins. Fc-fusion proteins are chimeric proteins consisting of the effector region of a protein, such as the Fab region of an antibody or the binding region of a receptor, fused to the Fc region of an immunoglobulin that is frequently an immunoglobulin G (IgG). Fc-fusion proteins are widely used as therapeutics as they offer advantages conferred by the Fc region, such as:                The possibility of purification using protein A or protein G affinity chromatography with affinities which vary according to the IgG isotype. Human IgG1, IgG2 and IgG4 bind strongly to Protein A and all human IgGs including IgG3 bind strongly to Protein G;        An increased half-life in the circulatory system, since the Fc region binds to the salvage receptor FcRn which protects from lysosomal degradation;        Depending on the medical use of the Fc-fusion protein, the Fc effector functions may be desirable. Such effector functions include antibody-dependent cellular cytotoxicity (ADCC) through interactions with Fc receptors (FcγRs) and complement-dependent cytotoxicity (CDC) by binding to the complement component 1q (C1q). IgG isoforms exert different levels of effector functions. Human IgG1 and IgG3 have strong ADCC and CDC effects while human IgG2 exerts weak ADCC and CDC effects. Human IgG4 displays weak ADCC and no CDC effects.        
Serum half-life and effector functions can be modulated by engineering the Fc region to increase or reduce its binding to FcRn, FcγRs and C1q respectively, depending on the therapeutic use intended for the Fc-fusion protein.
In ADCC, the Fc region of an antibody binds to Fc receptors (FcγRs) on the surface of immune effector cells such as natural killers and macrophages, leading to the phagocytosis or lysis of the targeted cells.
In CDC, the antibodies kill the targeted cells by triggering the complement cascade at the cell surface. IgG isoforms exert different levels of effector functions increasing in the order of IgG4<IgG2<IgG1=IgG3. Human IgG1 displays high ADCC and CDC, and is the most suitable for therapeutic use against pathogens and cancer cells.
Under certain circumstances, for example when depletion of the target cell is undesirable, abrogating effector functions is required. On the contrary, in the case of antibodies intended for oncology use, increasing effector functions may improve their therapeutic activity (Carter et al., 2006)
Modifying effector functions can thus be achieved by engineering the Fc region to either improve or reduce binding of FcγRs or the complement factors.
The binding of IgG to the activating (FcγRI, FcγRIIa, FcγRIIIa and FcγRIIIb) and inhibitory (FcγRIIb) FcγRs or the first component of complement (C1q) depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are critical for FcγRs and complement C1q binding, and have unique sequences in IgG2 and IgG4. For instance, substitution of IgG2 residues at positions 233-236 into human IgG1 greatly reduced ADCC and CDC (Armour et al., 1999 and Shields et al., 2001).
Numerous mutations have been made in the CH2 domain of IgG and their effect on ADCC and CDC was tested in vitro (Shields et al., 2001, Idusogie et al., 2001 and 2000, Steurer et al., 1995). In particular, a mutation to alanine at E333 was reported to increase both ADCC and CDC (Idusogie et al., 2001 and 2000).
Increasing the serum half-life of a therapeutic antibody is another way to improve its efficacy, allowing higher circulating levels, less frequent administration and reduced doses. This can be achieved by enhancing the binding of the Fc region to neonatal FcR (FcRn). FcRn, which is expressed on the surface of endothelial cells, binds the IgG in a pH-dependent manner and protects it from degradation. Several mutations located at the interface between the CH2 and CH3 domains have been shown to increase the half-life of IgG1 (Hinton et al., 2004 and Vaccaro et al., 2005).
The following Table 1 summarizes some known mutations of the IgG Fc-region (taken from Invivogen's website).
IgGEngineered FcIsotypeMutationsPropertiesPotential BenefitsApplicationshIgG1e1humanT250Q/M428LIncreasedImproved localizationVaccination;IgG1plasma half-to target; increasedtherapeuticlifeefficacy; reduced doseuseor frequency ofadministrationhIgG1e2humanM252Y/S254T/T256E +IncreasedImproved localizationVaccination;IgG1H433K/N434Fplasma half-to target; increasedtherapeutic uslifeefficacy; reduced doseor frequency ofadministrationhIgG1e3humanE233P/L234V/L235A/ReducedReduced adverseTherapeuticIgG1?G236 +ADCC andeventsuse withoutA327G/A3305/P331SCDCcell depletionhIgG1e4humanE333AIncreasedIncreased efficacyTherapeuticIgG1ADCC anduse with cellCDCdepletionhIgG2e1humanK322AReducedReduced adverseVaccination;IgG2CDCeventstherapeuticuse
Given the therapeutic utility of Fc-containing proteins, particularly antibodies and Fc-fusion proteins, there is a need for significant amounts of highly purified protein that is adequate for human administration. Effective purification processes are suitable for large-scale purification of Fc-containing proteins.